Clonal strains of Moloney and Rauscher murine leukemia viruses (M- and R-MuLV) have been demonstrated to induce tumors of T and B lymphoid cells, respectively. To investigate the basis for their cell specificity, these viruses have been cloned and analyzed for their molecular organization and attempts have been made to construct recombinant viruses for virologic testing. The physical map of unintegrated M-MuLV cloned in its Hind III site was consistent with that previously published. R-MuLV was cloned in its integrated form containing cellular flanking sequences at both ends. The cloned molecule is approximately 12.5 + 1.25 kbp in length and contains more than 9 kbp of viral DNA. The cloned DNA induced virus production in NIH/3T3 cells upon transfection. The p12 and gp70 proteins of virus released were shown to possess antigenic determinants specific for R-MuLV. Heteroduplex analysis showed complete homology between R-MuLV DNA and Rauscher 70S RNA or M-MuLV DNA. On the other hand, restriction maps showed a strong correlation of cloned R-MuLV DNA with unintegrated linear R-MuLV DNA but not with M-MuLV DNA.